Genetic transformation of cotton with a harpin-encoding gene hpaXoo confers an enhanced defense response against different pathogens through a priming mechanism

<p>Abstract</p> <p>Background</p> <p>The soil-borne fungal pathogen <it>Verticillium dahliae </it>Kleb causes <it>Verticillium </it>wilt in a wide range of crops including cotton (<it>Gossypium hirsutum</it>). To date, most upland cotton varieties are susceptible to <it>V. dahliae </it>and the breeding for cotton varieties with the resistance to <it>Verticillium </it>wilt has not been successful.</p> <p>Results</p> <p>Hpa1_Xoois a harpin protein from <it>Xanthomonas oryzae </it>pv. <it>oryzae </it>which induces the hypersensitive cell death in plants. When <it>hpa1</it>_{<it>Xoo </it>}was transformed into the susceptible cotton line Z35 through <it>Agrobacterium</it>-mediated transformation, the transgenic cotton line (T-34) with an improved resistance to <it>Verticillium dahliae </it>was obtained. Cells of the transgenic T-34, when mixed with the conidia suspension of <it>V. dahliae</it>, had a higher tolerance to <it>V. dahliae </it>compared to cells of untransformed Z35. Cells of T-34 were more viable 12 h after mixing with <it>V. dahliae </it>conidia suspension. Immunocytological analysis showed that Hpa1_Xoo, expressed in T-34, accumulated as clustered particles along the cell walls of T-34. In response to the infection caused by <it>V. dahliae</it>, the microscopic cell death and the generation of reactive oxygen intermediates were observed in leaves of T-34 and these responses were absent in leaves of Z35 inoculated with <it>V. dahliae</it>. Quantitative RT-PCR analysis indicated that five defense-related genes, <it>ghAOX1, hin1, npr1, ghdhg-OMT</it>, and <it>hsr203J</it>, were up-regulated in T-34 inoculated with <it>V. dahliae</it>. The up-regulations of these defense-relate genes were not observed or in a less extent in leaves of Z-35 after the inoculation.</p> <p>Conclusions</p> <p>Hpa1_Xooaccumulates along the cell walls of the transgenic T-34, where it triggers the generation of H₂O₂as an endogenous elicitor. T-34 is thus in a primed state, ready to protect the host from the pathogen. The results of this study suggest that the transformation of cotton with <it>hpa1</it>_{<it>Xoo </it>}could be an effective approach for the development of cotton varieties with the improved resistance against soil-borne pathogens.</p

Genetics and resistance/Génétique et résistance Development of EST-derived simple sequence repeat markers for wheat leaf rust fungus, Puccinia triticina Eriks Molecular markers for wheat leaf rust

Abstract: Gene-associated simple sequence repeat (SSR) markers were developed for Puccinia triticina through the data mining of existing EST libraries. Analysis of 7134 expressed sequence tags (ESTs) from cDNA libraries of P. triticina detected 204 EST-SSRs with a minimum of 12 repeating nucleotides. The majority of EST-SSRs contained short di-or tri-nucleotide repeats. These EST-SSRs were evaluated on 35 P. triticina isolates collected in Canada and 21 EST-SSRs were polymorphic and informative in determining intraspecific genetic diversity. A comparison of virulence and EST-SSR genotypes showed a strong correlation between virulence to Lr2a, Lr2c and Lr17a and EST-SSRs genotypes. The differentiation of the P. triticina population based on EST-SSR genotypes was comparable to that obtained with genomic SSRs, despite differences between two types of SSR markers. Eight of the 21 EST-SSRs produced the cross amplification in Puccinia coronata and Puccinia graminis, suggesting that EST-SSRs are more applicable than genomic SSRs for interspecific analysis. In summary, our study suggests that the data mining of EST databases is a feasible way to generate informative molecular markers for genetic studies of P. triticina. Keywords: population genetics, Puccinia triticina, simple sequence repeat, virulence Résumé: Des marqueurs microsatellites (SSR) associés à des gènes ont été conçus pour Puccinia triticina à partir l&apos;exploration de données contenues dans des banques d&apos;étiquettes de séquences exprimées (EST). L&apos;analyse de 7134 EST issues de banques d&apos;ADNc de P. triticina ont permis de détecter 204 EST-SSR avec un minimum de 12 répétitions de nucléotides. La majorité des EST-SSR contenaient de courtes répétitions di-ou tri-nucléotidiques. Ces EST-SSR ont été évaluées sur 35 isolats de P. triticina collectés au Canada. Parmi celles-ci, 21 étaient polymorphiques et ont fourni de l&apos;information servant à établir la diversité génétique intraspécifique. Une comparaison de la virulence et des génotypes EST-SSR a montré une forte corrélation entre la virulence à l&apos;égard de Lr2a, Lr2c et Lr17a ainsi qu&apos;à l&apos;égard des génotypes EST-SSR. La différenciation de la population de P. triticina, basée sur les génotypes EST-SSR, était comparable à celle obtenue avec les SSR génomiques, malgré les différences entre les deux types de marqueurs. Huit des 21 EST-SSR ont engendré la transférabilité chez Puccinia coronata et Puccinia graminis, ce qui suggère que les EST-SSR sont plus adaptables à l&apos;analyse interspécifique que les SSR génomiques. En résumé, notre étude suggère que l&apos;exploration de données des banques d&apos;EST permet de générer des marqueurs moléculaires informatifs pour les études génétiques portant sur P. triticina

Transcriptome analysis of Hpa1Xoo transformed cotton revealed constitutive expression of genes in multiple signalling pathways related to disease resistance

The transcriptome profile in leaves and roots of the transgenic cotton line T-34 expressing hpa1Xoo from Xanthomonas oryzae pv. oryzae was analysed using a customized 12k cotton cDNA microarray. A total of 530 cDNA transcripts involved in 34 pathways were differentially expressed in the transgenic line T-34, in which 123 differentially expressed genes were related to the cotton defence responses including the hypersensitive reaction, defence responses associated with the recognition of pathogen-derived elicitors, and defence signalling pathways mediated by salicylic acid, jasmonic acid, ethylene, auxin, abscicic acid, and Ca2+. Furthermore, transcripts encoding various leucine-rich protein kinases and mitogen-activated protein kinases were up-regulated in the transgenic line T-34 and expression of transcripts related to the energy producing and consuming pathway was also increased, which suggested that the enhanced metabolism related to the host defence response in the transgenic line T-34 imposed an increased energy demand on the transgenic plant

Development of a specific and reliable molecular marker to detect Stachybrotyrs [i.e. Stachybotrys] elegans, a destructive mycoparasite of Rhizoctonia solani

Stachybotrys elegans (Pidopl.) W. Gams is a destructive mycoparasite of the soilborne plant pathogen Rhizoctonia solani. It colonizes effectively all types of cells of R. solani, and is considered as an effective biological control agent (BCA). Monitoring the presence of this mycoparasite in the field trials requires the development of a reliable and sensitive diagnostic assay that is able to detect and differentiate the BCA from their target host. To achieve this, designed SCAR (sequenced characterized amplified regions) primers designated as SE-13F and SE-13R were generated from informative RAPD markers. They were tested in conventional PCR assays alone or in conjunction with the recently developed SCAR primers (SBU-177/336) designed for Rhizoctonia solani (Kuhn) on several types of DNA. These included DNA extracted from pure cultures, co-cultures of the BCA and the pathogen, plant tissue and several types of soils inoculated with both the BCA and the pathogen. Irrespective of the type of the biological samples from which the DNA was extracted, the primers SE-13F/SE-13R successfully amplified only S. elegans. No cross-reaction was observed when the primers were used to amplify DNA of other fungi, bacteria and plant tissues. Likewise, the primer pair SBU-177/336 detected only its target organism, i.e., R. solani. The detection limit using these primers on amplified DNA was as little as 1 pg DNA extracted from pure cultures of S. elegans, 100 pg DNA extracted from greenhouse soil and 33 pg DNA extracted from natural soil. This work is the first report on the development of SCAR markers for the BCA, S. elegans. These molecular markers offer not only an alternative diagnostic assay to conventional detection methods, but also the possibility of being used in ecological studies

Silver-Neodymium Codoped Lithium Aluminum Metaphosphate Glasses for Radio-Photoluminescence Dosimeter

Commercial radio-photoluminescence (RPL) glass dosimeters generally use Ag single-doped phosphate glass as a single-wavelength sensor. Now, a novel type of Ag–Nd-codoped phosphate glass has been developed, which can be applied to dual-wavelength or multi-wavelength RPL sensors, and can thus improve the accuracy and stability of RPL dosimeters. An anhydrous 99.5 (0.7LiPO3–0.3Al (PO3)3) −0.25Ag2O–0.25Nd2O3 glass was prepared and irradiated at different doses, and then the absorption, fluorescence, infrared transmission spectra, as well as fluorescence lifetimes were tested and analyzed. The results show that there is an energy transfer between the Ag defect center and Nd3+ ions, and the transfer efficiency using 380 nm excitation is greater than that using 310 nm excitation. Aside from the 650 nm fluorescence of the Ag defect center, strong 882 nm and 1054 nm fluorescences of Nd ions are exhibited. It is possible that these fluorescences would allow the developed Ag–Nd-codoped phosphate glass to be applied to new RPL glass sensors and dosimeters

Implications of Crop Rotation and Fungicide on Fusarium and Mycotoxin Spectra in Manitoba Barley, 2017&ndash;2019

Fusarium head blight (FHB) is one of the most important diseases of barley in Manitoba province (western Canada), and other major barley producing regions of the world. Little is known about the Fusarium species and mycotoxin spectra associated with FHB of barley in Manitoba. Hence, barley grain samples were collected from 149 commercial fields from 2017 to 2019, along with information on respective cropping history, and analyzed with respect to Fusarium species spectra, abundance, chemotype composition, and mycotoxin profiles. Fusarium poae was the predominant Fusarium species associated with FHB of barley in Manitoba, followed by F. graminearum, and F. sporotrichioides; F. equiseti and F. avenaceum were also detected but at low levels. F. poae strains with the nivalenol (NIV) chemotype and F. graminearum strains with 3-acetyl deoxynivalenol (3-ADON) and 15-acetyl deoxynivalenol (15-ADON) chemotypes were commonly detected in the barley grain samples. Nivalenol (597.7, 219.1, and 412.4 &micro;g kg&minus;1) and deoxynivalenol (DON) (264.7, 56.7, and 65.3 &micro;g kg&minus;1) were the two most prevalent mycotoxins contaminating Manitoba barley in 2017, 2018 and 2019, respectively. A substantially higher DON content was detected in grain samples from barley fields with cereals as a preceding crop compared to canola and flax. Furthermore, F. poae proved less sensitive to four triazole fungicides (metconazole, prothioconazole+tebuconazole, tebuconazole, and prothioconazole) than F. graminearum. Findings from this research will assist barley producers with improved understanding of FHB threat levels and optimizing practices for the best management of FHB in barley

Influence of Mixed Na2O/K2O on Chemical Durability and Spectral Properties of P2O5-Al2O3-BaO-K2O-Na2O-Nd2O3 Phosphate Glasses

A series of 56P2O5-7.5Al2O3-5.9BaO-(28.56-x)K2O-xNa2O-1.51Nd2O3 phosphate glasses with different Na/(Na+K) ratios, which were specially designed for high-power laser application, were prepared by a high-temperature melting method. Except for the density, refractive index, glass transition temperature, and DC conductivity, the chemical durability and spectral properties, as emphasized by high-power and high-energy laser material, were further measured and analyzed. Regarding the chemical durability, the dissolution rates of these glasses do not show an evident mixed alkali effect with increasing the Na/(Na+K) ratio, although the effect is obvious for the glass transition temperature and DC conductivity. To better understand the nature of the dissolution mechanism, the ionic release concentrations of every element are determined. Both Na and K undergo ion exchange, but the ion exchange rate of K is much larger than that of Na. In terms of the spectral properties, the J&ndash;O parameters, emission cross-section, radiation lifetime, fluorescence lifetime, effective bandwidth, fluorescence branching ratio, and quantum efficiency are determined from absorption and emission spectra. The trend of &Omega;2 deviating from linearity indicates that the coordination environment symmetry of Nd3+ ions and the covalence of Nd-O also present an evident mixed alkali effect. The most important finding is that the emission cross-section and fluorescence lifetime of Nd3+ ions at 1053 nm were not affected by the change in the Na/K ratio. According to the above experimental results, the optimized value of the Na/K ratio was determined, based on which the 56P2O5-7.5Al2O3-5.9BaO-(28.56-x)K2O-xNa2O-1.51Nd2O3 glass maintains a high emission cross-section with good chemical durability